The major aims of of this research proposal are to elucidate the role of the nucleus in influenza virus replication and to determine the effect of virus infection on some host nuclear functions. Four of the virus-specific proteins in the infected cell are associated with the nucleus, apparently primarily with the nucleolar fraction. To obtain a more complete analysis of this nuclear accumulation, we intend to: (1) characterize the two putative nonstructural proteins in the nucleus; (2) determine definitively the intranuclear localization of virus-specific proteins; (3) determine the time course during infection of the appearance of these proteins in the nucleus; (4) determine whether any of these proteins migrate back to the cytoplasm at any time of infection. In addition, we will determine: the effects of virus infection on the host nucleolar functions of ribosomal precursor RNA synthesis and processsing; and whether these effects provide an indirect assay for the activity of virus-specific products in the nucleus. We will determine whether these products inhibit ribosomal RNA synthesis catalyzed by whole nuclei in vitro and whether any viral temperature-sensitive (ts) mutants are ts in their ability to effect host ribosomal RNA synthesis. If such mutants are identified, we will analyze the consequences of such a ts defect in order to determine the virus-specific functions requiring nuclear events. We will also analyze the infective process in a mutant host cell, ts in a nucleolar function. To assess the possible role of the nucleus in the synthesis of virion RNA (vRNA), we intend: (1) to determine the sites of vRNA synthesis in the cell; (2) to identify ts mutants defective in vRNA synthesis and to determine whether this defect is reflected in a defect in the nuclear accumulation of virus-specific proteins; and (3) to purify and characterize the vRNA replicase.